Summary: Research on the seroepidemiology of the hepatitis B virus (HBV) and hepatitis C virus (HCV) in serially collected serum samples from Miyazaki, Japan, has continued. One of the villages has a 23% prevalence rate of HCV infection, and studies have shown that the incidence of NEW infections in previously seronegative persons is extremely high, approaching 300 cases/100,000 person-years. Later studies in other villages elsewhere in Japan published by other laboratories have subsequently provided additional support for the concept that pockets of extremely high prevalence of HCV are probably responsible for the high rate of HCV-associated serious liver disease in Japan. The quasispecies nature of the hepatitis C virus (HCV) is thought to play a central role in maintaining and modulating viral persistence. Changes in the components of this quasispecies population allow the virus to escape from the host immune response. Serial serum samples were obtained over a 27-year period from an HCV-infected patient and a nurse who became infected with HCV by a broken capillary pipette contaminated with the patient's blood. The effects on the quasispecies distribution caused by the immune globulin given to the nurse on the day of infection and four weeks later were examined. The hypervariable region 1 (HVR1) and 5'non-coding region (5'NCR) of the HCV genome were amplified from each serum sample by PCR and cloned. Twenty clones per sample for the HVR1 gene and five clones per sample for the 5'NCR gene were sequenced and analyzed. All serum samples contained a heterogeneous mixture of HCV quasispecies. The HVR1 sequence was more diversified among the 4 serum samples obtained from the patient (80 clones) than among the 12 serum samples from the nurse (240 clones), as indicated by a two-fold higher mean genetic distance in the patient (16.64) compared to the nurse (8.78). All HVR1 sequences from both the patient and the nurse had more synonymous substitutions than non-synonymous substitutions. In contrast, the 5'NCR sequence was highly conserved; three nucleotide substitutions were found among of a total of 80 clones of this 256 nucleotide fragment (five clones from each of 16 specimens). In the first serum specimen from the patient and the first two serum specimens from the nurse, most of the 20 clones from each serum sample had one common sequence in the HVR1 gene. The uniformity of the HVR1 sequence in these samples and the emergence of greater diversity in later serum samples is consistent with the apparent transmission of HCV between the patient and the nurse and clarifies the pattern of quasispecies diversity in an individual over time.